| Product Name |
WB-Validated SEMA4D Knockdown Cell Lysate Kit |
| Aliases |
SEMA4D; Semaphorin 4D; CD100; C9orf164; Coll-4; SEMAJ; Sema Domain, Immunoglobulin Domain (Ig), Transmembrane Domain (TM) And Short Cytoplasmic Domain, (Semaphorin) 4D; Semaphorin-4D; FLJ39737; BB18; GR3; A8; Sema Domain, Immunoglobulin Domain (Ig), Transmembrane Domain (TM) And Short Cytoplasmic Domain, 4D; Chromosome 9 Open Reading Frame 164; CD100 Antigen; M-Sema G; M-Sema-G; COLL4 |
| Background |
Gene Name: SEMA4D
NCBI Gene Entry:
10507
|
| Storage |
Cell Lysate: Store at -80°C for one year.
Antibody: Store at -20 °C for one year. |
| Kit Components |
1. WB-validated SEMA4D Knockdown Cell Lysate (100 μg )
2. WT Cell Lysate (100 μg )
3. Verification Tool: KD-Validated Semaphorin 4D Recombinant Rabbit mAb #61975 (5 μL) |
| Tested Cell Line |
HeLa |
| Validation Methods |
RT-qPCR; Western Blotting (WB) |
| Shipping |
Cell Lysate: Shipped with dry ice. Immediately store the product in a standard freezer at -80°C upon receipt.
Antibody: Shipped with dry ice. Immediately store the product at -20°C upon receipt. |
| Instructions For Use |
This knockdown cell lysate should be paired with wild-type HeLa cell lysate for use.
Tips: 1. For reducing SDS-PAGE, add DTT to 50–100 mM final or β-mercaptoethanol to 2–5% final (v/v), plus 0.01-0.1% bromophenol blue. Heat at 95°C for 5 minutes, cool on ice, and load the samples.
2. For non-reducing SDS-PAGE, add bromophenol blue to a final concentration of 0.01-0.1%. Do not add DTT or β-mercaptoethanol. Heat at 70°C for 5–10 minutes before loading. |
| Manufacturing Process |
The following protocol was used to generate mRNA knockdown cell lysate:
1. Release 0.5 million HeLa cells into a 35 mm tissue culture dish in 2 mL of the growth medium (DMEM containing 10% FBS and 1% pen/strep). Cell density should reach 50-60% confluence the following day.
2. 24 h after cell release, pre-warm the shRNA lentiviral medium to 37°C.
3. Discard 1 mL of the original growth medium of the 35 mm dish.
4. Using a serological pipette, gently mix the lentiviral solution 3 times.
5. Carefully add 1 mL of the lentiviral solution to the well. Tip: To prevent splashing, add the solution to the dish along the wall.
6. Add a polybrene stock solution to the culture medium at a final concentration of 5 μg/mL. Gently swirl the dish to mix.
7. 48 h after cell release, without discarding the original medium, add another 1 mL of lentiviral medium directly into the dish.
8. Add an additional polybrene stock solution into the dish to obtain a final concentration of 5 μg/mL. Tip: Now, the medium in the dish should be a total of 3 mL.
9. 72 h after cell release, cells may reach confluence. Trypsinize the cells off the 35 mm dish and culture those cells in a 60 mm dish.
10. Add puromycin to the dish at a final concentration of 4 μg/mL. Tip: To assess the efficacy of puromycin selection, culture a dish of wild-type HeLa cells as a negative control.
11. Allow puromycin selection for 48 h. Almost all wild-type HeLa cells should die, while the dish infected with lentiviruses should have some remaining cells.
12. Replace the medium with regular growth medium without puromycin and allow the cells to grow to confluence before harvesting or staining.
13. Cells were lysed with IntactProtein™ Universal Protein Extraction Kit (Cat#415) and stored in -80℃. |
| Note |
This product is for research use only. |
